Journal of Obstetrics and Gynaecology, 2014; Early Online: 1–4 © 2014 Informa UK, Ltd. ISSN 0144-3615 print/ISSN 1364-6893 online DOI: 10.3109/01443615.2014.937330
Spontaneous abortion and functional polymorphism (Val16Ala) in the manganese SOD gene E. Eskafi Sabet1, Z. Salehi1, S. Khodayari2, S. Sabouhi Zarafshan1 & Z. Zahiri3 1Department of Biology, Faculty of Sciences, University of Guilan, 2Department of Biology, International Pardis, University of Guilan and 3Reproductive Health Research Center, Obstetrics and Gynaecology Department, Alzahra Hospital, Guilan University of Medical Sciences,
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Rasht, Iran
Spontaneous abortion is the most common complication of early pregnancy. Genetic factors have been hypothesised to play a role in spontaneous abortion. Since it is possible that the balance of oxidants and antioxidants can be affected by different genetic variants, gene polymorphisms have been proposed as a susceptibility factor that increases the chance of abortion. Manganese superoxide dismutase is an important antioxidant enzyme encoded by manganese superoxide dismutase (MnSOD) gene. The aim of this experiment was to assess whether Val16Ala polymorphism of MnSOD gene is associated with abortion in north of Iran. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used for genotyping. Statistical analyses were conducted using the c2-test. The genetic distributions did not differ significantly between cases and controls, however slightly more Val/Val genotypes were found among the patients compared with control subjects (p 0.059). No correlation was observed between susceptibility to abortion and MnSOD Val16Ala polymorphism. Larger population-based studies are needed for clarifying the relationship between abortion and MnSOD genotypes. Keywords: Gene polymorphism, manganese superoxide dismutase, reactive oxygen species, spontaneous abortion
Introduction Spontaneous abortion, usually called miscarriage, is the most common complication of early pregnancy, where a pregnancy ends with the death of the fetus prior to 20 weeks or with a fetus born weighing 500 g (WHO 1976). About 15–20% of pregnancies end in miscarriage (Demetroulis et al. 2001). Miscarriages are caused by a variety of factors, including polycystic ovary syndrome, uncontrolled diabetes, infection, thrombophilia, genetics, abnormalities of the uterus, accidental trauma and alcohol consumption (Ruutiainen and Seppala 1991; Homer et al. 2000; Sarig et al. 2002; Hefler et al. 2002; Todorova et al. 2005; McDonald et al. 1992). Apart from the above factors, the aetiology of miscarriage remains unclear in about 40% of the cases (Hempstock et al. 2003). In one hypothesis, oxidative stress is suggested as a main mechanism underlying the pathogenesis of a continuum of disease processes, such as spontaneous abortion, hydatidiform mole and pre-eclampsia (Gupta et al. 2007). Reactive oxygen species (ROS) are activated molecules generated through aerobic respiration and oxygen metabolism. ROS are involved in the physiology
of the female reproductive system. They serve as the key signal molecules in physiological processes involving the female reproductive tract from oocyte maturation to fertilisation, such as folliculogenesis, oocyte maturation, corpus luteum and uterine function, embryogenesis, embryonic implantation and fetoplacental development (Agarwal et al. 2008). Control of ROS generation by antioxidant system is an important process for normal pregnancy (Vural et al. 2000). Manganese superoxide dismutase (MnSOD) is a mitochondrial antioxidant enzyme that protects against ROS. MnSOD is translated in the cytoplasm and then imported into mitochondria, where it catalyses the conversion of superoxide anion (O2–) to hydrogen peroxide (H2O2). The superoxide radical is the primary ROS formed when electrons leak from the electron transport chain (Halliwell et al. 1992). The human MnSOD gene is located on chromosome 6 (6q25.3) and its transcription site is preceded by a GC rich promoter region containing a cluster of seven SP1 and three AP2 sequences with no TATA or CAAT boxes (Wan et al. 1994). Among genetic polymorphisms in the MnSOD gene, Val16Ala in a mitochondrial target sequence (MTS) have been known as the most common functional polymorphism (rs4880). This single nucleotide polymorphism (SNP) is caused by a single T to C substitution (Rosenblum et al. 1996). Computer models predicted that the T allele would encode a beta-sheet conformation of the MnSOD, leading to retention of the Val variant in the mitochondrial membrane. However, the alpha-helical structure of the Ala-containing MnSOD shows 30–40% more efficient transport and localisation to the mitochondrial matrix (Shimoda-Matsubayashi et al. 1996; Sutton et al. 2003). To date, the relationship between Val16Ala polymorphism in the MnSOD gene and the risk of abortion has not been evaluated. The aim of this study was the analysis of MnSOD gene in women with spontaneous abortion in the north of Iran.
Materials and methods Sampling The characteristics of the samples are shown in Table I. Clinical data, including age at the time of abortion, number of abortions, number of live births, history of medications and disease backgrounds, to name only a few, were recorded for all the samples. Inclusion criteria were non-pregnant women without any specific disease, having the history of at least one spontaneous abortion. Excluded criteria were: chromosomal abnormalities, reproductive diseases, maternal endocrine and immune
Correspondence: Z. Salehi, Department of Biology, Faculty of sciences, University of Guilan, Rasht, Iran. E-mail: [email protected]
2 E. Eskafi Sabet et al. Table I. Characteristics of the samples. Women with abortion (n 100) n
Characteristics Maternal age (years) (range, mean) Body mass index (range, mean) Length of menstrual cycle (days) (range, mean) Family history of miscarriage Daily ETS Number of live births 0 1 2 3
18–44 (28.8) 18.4–29.3 (23.5) 28–30 (28.5) 33 15 61 33 6 –
Controls (n 79)
(%)
n
(%)
33 15
20–40 (27.4) 19.0–27.6 (24.0) 27–33 (28.7) 14 9
17.7 11.3
– 25 39 15
31.6 49.3 18.9
61 33 6
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ETS, exposure to environmental tobacco smoke.
disorders, maternal coagulation defects, hormonal treatment, irregular menstrual cycle and anti-inflammatory drugs. All the participants resided in Guilan province and attended the study between August 2011 and January 2012. Informed consent was obtained from all the subjects and ethical principles of the Helsinki Declaration were followed.
DNA extraction For each individual, blood samples (2 ml) were collected by venipuncture and drawn into heparin-containing tubes (Venoject, Belgium). Genomic DNAs were extracted from peripheral leukocytes using DNG-plus DNA extraction kit (CinnaGen, Iran). DNA concentrations were quantified with a spectrophotometer and extracted DNAs were stored at –20°C until further analysis.
Genotyping The MnSOD fragment was amplified using polymerase chain reaction (PCR) and the Val16Ala polymorphism was genotyped by restriction fragment length polymorphism (RFLP) technique. PCR amplification was carried out in a total volume of 25 ml containing 30 ng genomic DNA, 1 PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 0.5 mM each designed primer (forward, 5′-CGGGCTGTGCTTTCTCGTC-3′; reverse, 5′TCAGCCTGGAACCTACCCTT-3′) and 1.5 unit of super Taq DNA polymerase (Gen Fanavaran, Iran). The thermal profile involved initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 58°C for 60 s, extension at 72°C for 45 s and completed with a final extension at 72°C for 5 min. The 243 bp PCR product was digested with 1 unit of BSaWІ (New England Biolabs, Beverly, MA) in a total volume of 10 ml containing 1 buffer 4, 1 BSA and 0.1 mg DNA. The reaction mixture was incubated at 60°C for 1 h. The digested products were separated on 3% agarose gel stained with ethidium bromide and visualised under UV light. Samples homozygote for Ala/Ala genotype revealed a 243 bp fragment. Individuals’ heterozygote for Val/Ala genotype resulted in three 243, 196 and 47 bp fragments. Samples with Val/Val homozygote genotype produced two 196 and 47 bp fragments. All the samples with ambiguous results were re-genotyped for quality control purposes.
by chance were 5% (p 0.05). All statistical analyses were conducted using MedCalc statistical software (Version 12.1, Mariakerke, Belgium).
Results The patients were 24 women with one spontaneous abortion, 46 women with two and 30 women with more than three abortions, aged from 18 to 44 years old (mean 28.8). A total of 20% of miscarriages occurred before 8 weeks’ gestation, 33.4% at 9–12 and 45.6% at 13–20 weeks. The controls were 79 women having normal pregnancy history aged from 20 to 40 years old (mean 27.4). There was no significant difference in body mass index (BMI), exposure to environmental tobacco smoke (ETS), family history of miscarriage and age distributions, between cases and controls. The genotypes of MnSOD were identified by PCR-RFLP (Figures 1 and 2). Table II shows the genotype and allele frequencies of MnSOD Val16Ala polymorphism among patients and controls. Among women with abortion, genotype frequencies were 20% for Val/Val, 76% for Val/Ala, and 4% for Ala/Ala. Among the control subjects, 12.6% were Val/Val, 74.7% were Val/Ala and 12.6% were Ala/Ala. We found no significant difference in genotype distributions between cases and controls, however slightly more Val/Val genotypes were observed among the patients compared with control subjects (p 0.059). Allele frequencies were 58% for Val and 42% for Ala in the patient group, and 50% for Val and 50% for Ala in the control group. No significant difference was found in allelic distributions between the two groups (p 0.160). In another analysis, the genotype distributions among the abortive group did not
Statistical analysis Genotype and allele frequencies were calculated in each group. The differences in genetic distributions between patients and controls were estimated by the c2-test. Results were considered statistically significant when the probability of findings occurring
Figure 1. Agarose gel electrophoresis after PCR. L: 50-bp ladder; 1, 2, 3, 4 shows the 243 bp fragment of MnSOD.
Spontaneous abortion and functional polymorphism (Val16Ala) in the manganese sod gene 3 Table III. Distribution of MnSOD Val16Ala polymorphisms in mono and poly-abortive patients. Mono-abortive
Figure 2. Agarose gel electrophoresis after RFLP Lanes: L, 50-bp ladder; 1, 2 Ala/Ala; 3, 4 Ala/Val; 5, 6 Val/Val.
differ significantly between the mono- and poly-abortive patients (p 0.2) (Table III).
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Discussion To our knowledge, this is the first report evaluating the association between MnSOD Val16Ala polymorphism and the risk of spontaneous abortion. MnSOD is a crucial enzyme counteracting ROS generated in mitochondria. Since the efficient targeting of MnSOD may be an important part of its functional activity and ROS contribute to aetiology of the disease, we performed a case–control study to examine the association between MnSOD genotypes and the risk of spontaneous abortion. The high metabolic rate of placenta leads to increased generation of ROS. As a result, oxidative stress rises during early pregnancy (Safronova et al. 2003). Increased formation of ROS, and even oxidative stress, is considered normal during the 2nd trimester of pregnancy (Wang et al. 1991). However, uncontrolled oxidative stress has been found to be associated with miscarriage during the establishment of cellular and biochemical interactions between the uterine endometrium and the developing post-implanting conceptus (Jenkins et al. 2000). Lyu et al. (2013) found that overproduction of ROS along with downregulating of some genes associated with mitochondrial functions, such as copper/zinc superoxide dismutase (Cu/Zn SOD), would lead to unexplained miscarriage resulting from abnormal responses in placental villi. Adequate placental antioxidant defence may prevent the disorders induced by oxidative stress, including impairment of placental function and abortion (Al-Gubory et al. 2010). The mitochondrial MnSOD is critically required because as much as 90% of cellular ROS can be generated in this organelle. In addition, mitochondrial DNA is particularly susceptible to oxidative damage (Richter et al. 1988). In this study, no significant difference was found between MnSOD Val16Ala polymorphism and the risk of spontaneous abortion. However, more Val/Val genotypes were observed among the patients. Our results were consistent with some previous studies, which found no association Table II. Distribution of MnSOD Val16Ala polymorphisms in women with spontaneous abortion (cases) and women with normal pregnancy history (controls). Controls
Genotype frequency Val/Val Val/Ala Ala/Ala Allele frequency Val Ala
Cases
n
(%)
n
(%)
10 59 10
12.6 74.7 12.6
20 76 4
20.0 76.0 4.0
79 79
50.0 50.0
116 84
58.0 42.0
p value 0.059
0.16
Genotype frequency Val/Val Val/Ala Ala/Ala Allele frequency Val Ala
Poly-abortive
n
(%)
n
(%)
3 21 0
12.5 87.5 0
17 55 4
22.3 72.3 5.2
28 21
56 43
89 63
58 41
p value 0.25
0.9
between MnSOD Val16Ala polymorphism and diseases, such as rheumatoid arthritis (Yen et al. 2002), asthma (Holla et al. 2005) and schizophrenia (Ventriglia et al. 2006). Association studies regarding the MnSOD Val16Ala polymorphism are difficult to interpret. As MnSOD removes the superoxide anion, a potential source of DNA damage, it would be plausible to predict that the MnSOD Val allele would lead to an increased risk of the disease. However, it has not been demonstrated strongly, as several association studies examining the relationship of this polymorphism with diseases, such as breast cancer, had adverse results (Mitrunen et al. 2001; Egan et al. 2003). The role of MnSOD polymorphism on some non-cancerous conditions, such as carotid atherosclerosis (Kakko et al. 2003), Alzheimer (Wiener et al. 2007) and pregnancy rates in IVF (Ruiz-Sanz et al. 2011), also showed contrasting results. In conclusion, no correlation was observed between susceptibility to abortion and MnSOD Val16Ala polymorphism. The effects of MnSOD genotypes may depend on environmental factors that increase or decrease the levels of ROS, and such factors may vary in different populations. Further studies in larger populations are needed to discover the role of this gene polymorphism on spontaneous abortion.
Acknowledgements The authors thank all people in the Genetics and Developmental Biology laboratories, Department of Biology, Faculty of Sciences, University of Guilan, for their technical assistance, and all the sample donors for their collaboration. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was partly supported by the University of Guilan.
References Agarwal A, Gupta S, Sekhon L, Shah R. 2008. Redox considerations in female reproductive function and assisted reproduction: from molecular mechanisms to health implications. Antioxidants and Redox Signalling 10:1375–1403. Al-Gubory KH, Fowler PA, Garrel C. 2010. The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. International Journal of Biochemistry and Cell Biology 42:1634–1650. Demetroulis C, Saridogan E, Kunde D, Naftalin AA. 2001. A prospective randomized control trial comparing medical and surgical treatment for early pregnancy failure. Human Reproduction 16:365–369. Egan KM, Thompson PA, Titus-Ernstoff L, Moore JH, Ambrosone CB. 2003. MnSOD polymorphism and breast cancer in a population-based case-control study. Cancer Letters 199:27–33.
J Obstet Gynaecol Downloaded from informahealthcare.com by Universitaet Zuerich on 01/06/15 For personal use only.
4 E. Eskafi Sabet et al. Gupta S, Agarwal A, Banerjee J, Alvarez JG. 2007. The role of oxidative stress in spontaneous abortion and recurrent pregnancy loss: a systematic review. Obstetrical and Gynecological Survey 62:335–347; quiz 353–354. Halliwell B, Gutteridge JM, Cross CE. 1992. Free radicals, antioxidants, and human disease: where are we now? Journal of Laboratory and Clinical Medicine 119:598–620. Hefler LA, Tempfer CB, Bashford MT, Unfried G, Zeillinger R, Schneeberger C et al. 2002. Polymorphisms of the angiotensinogen gene, the endothelial nitric oxide synthase gene, and the interleukin-1beta gene promoter in women with idiopathic recurrent miscarriage. Molecular Human Reproduction 8:95–100. Hempstock J, Jauniaux E, Greenwold N, Burton GJ. 2003. The contribution of placental oxidative stress to early pregnancy failure. Human Pathology 34:1265–1275. Holla LI, Kankova K, Vasku A. 2005. Functional polymorphism in the manganese superoxide dismutase. Clinical Biochemistry 39:299–309. Homer H, Li T, Cooke I. 2000. The septate uterus: a review of management and reproductive outcome. Fertility and Sterility 73:1–14. Jenkins C, Wilson R, Roberts J, Miller H, McKillop JH, Walker JJ. 2000. Antioxidants: their role in pregnancy and miscarriage. Antioxidants and Redox Signalling 2:623–628. Kakko S, Paivansalo M, Koistinen P, Kesaniemi YA, Kinnula VL, Savolainen MJ. 2003. The signal sequence polymorphism of the MnSOD gene is associated with the degree of carotid atherosclerosis. Atherosclerosis 168:147–152. Lyu SW, Song H, Yoon JA, Chin MU, Sung SR, Kim YS et al. 2013. Transcriptional profiling with a pathway-oriented analysis in the placental villi of unexplained miscarriage. Placenta 34:133–140. McDonald AD, Armstrong BG, Sloan M. 1992. Cigarette, alcohol, and coffee consumption and prematurity. American Journal of Public Health 82:87–90. Mitrunen K, Sillanpaa P, Kataja V, Eskelinen M, Kosma VM, Benhamou S et al. 2001. Association between manganese superoxide dismutase (MnSOD) gene polymorphism and breast cancer risk. Carcinogenesis 22:827–829. Richter C, Park JW, Ames BN. 1988. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proceedings of the National Academy of Sciences of the United States of America 85:6465–6467. Rosenblum J, Gilula N, Lerner R. 1996. On signal sequence polymorphisms and diseases of distribution. Proceedings of the National Academy of Sciences of the United States of America 93:4471–4473. Ruiz-Sanz JI, Aurrekoetxea I, Matorras R, Ruiz-Larrea MB. 2011. Ala16Val SOD2 polymorphism is associated with higher pregnancy rates in in vitro fertilization cycles. Fertility and Sterility 95:1601–1605.
Ruutiainen K, Seppala M. 1991. Polycystic ovary syndrome: evolution of a concept. Current Opinion in Obstetrics and Gynecology 3:326–335. Safronova VG, Matveeva NK, Avkhacheva NV, Sidel’nikova VM, Van’ko LV, Sukhikh GT. 2003. Changes in regulation of oxidase activity of peripheral blood granulocytes in women with habitual abortions. Bulletin of Experimental Biology and Medicine 136:257–260. Sarig G, Younis J, Hoffman R. 2002. Thrombophilia is common in women with idiopathic pregnancy loss and is associated with late pregnancy wastage. Fertility and Sterility 77:342–347. Shimoda-Matsubayashi S, Matsumine H, Kobayashi T, Nakagawa-Hattori Y, Shimizu Y, Mizuno Y. 1996. Structural dimorphism in the mitochondrial targeting sequence in the human manganese superoxide dismutase gene. A predictive evidence for conformational change to influence mitochondrial transport and a study of allelic association in Parkinson’s disease. Biochemical and Biophysical Research Communications 226:561–565. Sutton A, Khoury H, Prip-Buus C, Cepanec C, Pessayre D, Degoul F. 2003. The Ala16Val genetic dimorphism modulates the import of human manganese superoxide dismutase into rat liver mitochondria. Pharmacogenetics 13:145–157. Todorova K, Ivanov S, Mazneikova V, Genova M. 2005. Glucooxidative stress and spontaneous abortion in pregnant women with diabetes mellitus type 1. Akusherstvo i Ginekologiia 44:3–10. Ventriglia M, Scassellati C, Bonvicini C, Squitti R, Bevacqua MG, Foresti G et al. 2006. No association between Ala9Val functional polymorphism of MnSOD gene and schizophrenia in a representative Italian sample. Neuroscience Letters 410:208–211. Vural P, Akgul C, Yildirim A, Canbaz M. 2000. Antioxidant defence in recurrent abortion. Clinica Chimica Acta 295:169–177. Wan XS, Devalaraja MN, ST Clair DK. 1994. Molecular structure and organization of the human manganese superoxide dismutase gene. DNA and Cell Biology 13:1127–1136. Wang YP, Walsh SW, Guo JD, Zhang JY. 1991. Maternal levels of prostacyclin, thromboxane, vitamin E, and lipid peroxides throughout normal pregnancy. American Journal of Obstetrics and Gynecology 165:1690–1694. WHO. 1976. World Health Organization recommended definitions, terminology and format for statistical tables related to the perinatal period and use of a new certificate for cause of perinatal deaths. Acta Obstetricia et Gynecologica Scandinavica 56:247–253. Wiener HW, Perry RT, Chen Z, Harrell LE, Go RC. 2007. A polymorphism in SOD2 is associated with development of Alzheimer’s disease. Genes, Brain, and Behavior 6:770–775. Yen JH, Chen CJ, Tsai WC, Lin CH, Ou TT, Hu CJ et al. 2002. Manganese superoxide dismutase and cytochrome P450 1A1 genes polymorphisms in rheumatoid arthritis in Taiwan. Human Immunology 64:366–373.
Spontaneous abortion and functional polymorphism (Val16Ala) in the manganese SOD gene E. Eskafi Sabet1, Z. Salehi1, S. Khodayari2, S. Sabouhi Zarafshan1 & Z. Zahiri3 1Department of Biology, Faculty of Sciences, University of Guilan, 2Department of Biology, International Pardis, University of Guilan and 3Reproductive Health Research Center, Obstetrics and Gynaecology Department, Alzahra Hospital, Guilan University of Medical Sciences,
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Rasht, Iran
Spontaneous abortion is the most common complication of early pregnancy. Genetic factors have been hypothesised to play a role in spontaneous abortion. Since it is possible that the balance of oxidants and antioxidants can be affected by different genetic variants, gene polymorphisms have been proposed as a susceptibility factor that increases the chance of abortion. Manganese superoxide dismutase is an important antioxidant enzyme encoded by manganese superoxide dismutase (MnSOD) gene. The aim of this experiment was to assess whether Val16Ala polymorphism of MnSOD gene is associated with abortion in north of Iran. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used for genotyping. Statistical analyses were conducted using the c2-test. The genetic distributions did not differ significantly between cases and controls, however slightly more Val/Val genotypes were found among the patients compared with control subjects (p 0.059). No correlation was observed between susceptibility to abortion and MnSOD Val16Ala polymorphism. Larger population-based studies are needed for clarifying the relationship between abortion and MnSOD genotypes. Keywords: Gene polymorphism, manganese superoxide dismutase, reactive oxygen species, spontaneous abortion
Introduction Spontaneous abortion, usually called miscarriage, is the most common complication of early pregnancy, where a pregnancy ends with the death of the fetus prior to 20 weeks or with a fetus born weighing 500 g (WHO 1976). About 15–20% of pregnancies end in miscarriage (Demetroulis et al. 2001). Miscarriages are caused by a variety of factors, including polycystic ovary syndrome, uncontrolled diabetes, infection, thrombophilia, genetics, abnormalities of the uterus, accidental trauma and alcohol consumption (Ruutiainen and Seppala 1991; Homer et al. 2000; Sarig et al. 2002; Hefler et al. 2002; Todorova et al. 2005; McDonald et al. 1992). Apart from the above factors, the aetiology of miscarriage remains unclear in about 40% of the cases (Hempstock et al. 2003). In one hypothesis, oxidative stress is suggested as a main mechanism underlying the pathogenesis of a continuum of disease processes, such as spontaneous abortion, hydatidiform mole and pre-eclampsia (Gupta et al. 2007). Reactive oxygen species (ROS) are activated molecules generated through aerobic respiration and oxygen metabolism. ROS are involved in the physiology
of the female reproductive system. They serve as the key signal molecules in physiological processes involving the female reproductive tract from oocyte maturation to fertilisation, such as folliculogenesis, oocyte maturation, corpus luteum and uterine function, embryogenesis, embryonic implantation and fetoplacental development (Agarwal et al. 2008). Control of ROS generation by antioxidant system is an important process for normal pregnancy (Vural et al. 2000). Manganese superoxide dismutase (MnSOD) is a mitochondrial antioxidant enzyme that protects against ROS. MnSOD is translated in the cytoplasm and then imported into mitochondria, where it catalyses the conversion of superoxide anion (O2–) to hydrogen peroxide (H2O2). The superoxide radical is the primary ROS formed when electrons leak from the electron transport chain (Halliwell et al. 1992). The human MnSOD gene is located on chromosome 6 (6q25.3) and its transcription site is preceded by a GC rich promoter region containing a cluster of seven SP1 and three AP2 sequences with no TATA or CAAT boxes (Wan et al. 1994). Among genetic polymorphisms in the MnSOD gene, Val16Ala in a mitochondrial target sequence (MTS) have been known as the most common functional polymorphism (rs4880). This single nucleotide polymorphism (SNP) is caused by a single T to C substitution (Rosenblum et al. 1996). Computer models predicted that the T allele would encode a beta-sheet conformation of the MnSOD, leading to retention of the Val variant in the mitochondrial membrane. However, the alpha-helical structure of the Ala-containing MnSOD shows 30–40% more efficient transport and localisation to the mitochondrial matrix (Shimoda-Matsubayashi et al. 1996; Sutton et al. 2003). To date, the relationship between Val16Ala polymorphism in the MnSOD gene and the risk of abortion has not been evaluated. The aim of this study was the analysis of MnSOD gene in women with spontaneous abortion in the north of Iran.
Materials and methods Sampling The characteristics of the samples are shown in Table I. Clinical data, including age at the time of abortion, number of abortions, number of live births, history of medications and disease backgrounds, to name only a few, were recorded for all the samples. Inclusion criteria were non-pregnant women without any specific disease, having the history of at least one spontaneous abortion. Excluded criteria were: chromosomal abnormalities, reproductive diseases, maternal endocrine and immune
Correspondence: Z. Salehi, Department of Biology, Faculty of sciences, University of Guilan, Rasht, Iran. E-mail: [email protected]
2 E. Eskafi Sabet et al. Table I. Characteristics of the samples. Women with abortion (n 100) n
Characteristics Maternal age (years) (range, mean) Body mass index (range, mean) Length of menstrual cycle (days) (range, mean) Family history of miscarriage Daily ETS Number of live births 0 1 2 3
18–44 (28.8) 18.4–29.3 (23.5) 28–30 (28.5) 33 15 61 33 6 –
Controls (n 79)
(%)
n
(%)
33 15
20–40 (27.4) 19.0–27.6 (24.0) 27–33 (28.7) 14 9
17.7 11.3
– 25 39 15
31.6 49.3 18.9
61 33 6
J Obstet Gynaecol Downloaded from informahealthcare.com by Universitaet Zuerich on 01/06/15 For personal use only.
ETS, exposure to environmental tobacco smoke.
disorders, maternal coagulation defects, hormonal treatment, irregular menstrual cycle and anti-inflammatory drugs. All the participants resided in Guilan province and attended the study between August 2011 and January 2012. Informed consent was obtained from all the subjects and ethical principles of the Helsinki Declaration were followed.
DNA extraction For each individual, blood samples (2 ml) were collected by venipuncture and drawn into heparin-containing tubes (Venoject, Belgium). Genomic DNAs were extracted from peripheral leukocytes using DNG-plus DNA extraction kit (CinnaGen, Iran). DNA concentrations were quantified with a spectrophotometer and extracted DNAs were stored at –20°C until further analysis.
Genotyping The MnSOD fragment was amplified using polymerase chain reaction (PCR) and the Val16Ala polymorphism was genotyped by restriction fragment length polymorphism (RFLP) technique. PCR amplification was carried out in a total volume of 25 ml containing 30 ng genomic DNA, 1 PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 0.5 mM each designed primer (forward, 5′-CGGGCTGTGCTTTCTCGTC-3′; reverse, 5′TCAGCCTGGAACCTACCCTT-3′) and 1.5 unit of super Taq DNA polymerase (Gen Fanavaran, Iran). The thermal profile involved initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 58°C for 60 s, extension at 72°C for 45 s and completed with a final extension at 72°C for 5 min. The 243 bp PCR product was digested with 1 unit of BSaWІ (New England Biolabs, Beverly, MA) in a total volume of 10 ml containing 1 buffer 4, 1 BSA and 0.1 mg DNA. The reaction mixture was incubated at 60°C for 1 h. The digested products were separated on 3% agarose gel stained with ethidium bromide and visualised under UV light. Samples homozygote for Ala/Ala genotype revealed a 243 bp fragment. Individuals’ heterozygote for Val/Ala genotype resulted in three 243, 196 and 47 bp fragments. Samples with Val/Val homozygote genotype produced two 196 and 47 bp fragments. All the samples with ambiguous results were re-genotyped for quality control purposes.
by chance were 5% (p 0.05). All statistical analyses were conducted using MedCalc statistical software (Version 12.1, Mariakerke, Belgium).
Results The patients were 24 women with one spontaneous abortion, 46 women with two and 30 women with more than three abortions, aged from 18 to 44 years old (mean 28.8). A total of 20% of miscarriages occurred before 8 weeks’ gestation, 33.4% at 9–12 and 45.6% at 13–20 weeks. The controls were 79 women having normal pregnancy history aged from 20 to 40 years old (mean 27.4). There was no significant difference in body mass index (BMI), exposure to environmental tobacco smoke (ETS), family history of miscarriage and age distributions, between cases and controls. The genotypes of MnSOD were identified by PCR-RFLP (Figures 1 and 2). Table II shows the genotype and allele frequencies of MnSOD Val16Ala polymorphism among patients and controls. Among women with abortion, genotype frequencies were 20% for Val/Val, 76% for Val/Ala, and 4% for Ala/Ala. Among the control subjects, 12.6% were Val/Val, 74.7% were Val/Ala and 12.6% were Ala/Ala. We found no significant difference in genotype distributions between cases and controls, however slightly more Val/Val genotypes were observed among the patients compared with control subjects (p 0.059). Allele frequencies were 58% for Val and 42% for Ala in the patient group, and 50% for Val and 50% for Ala in the control group. No significant difference was found in allelic distributions between the two groups (p 0.160). In another analysis, the genotype distributions among the abortive group did not
Statistical analysis Genotype and allele frequencies were calculated in each group. The differences in genetic distributions between patients and controls were estimated by the c2-test. Results were considered statistically significant when the probability of findings occurring
Figure 1. Agarose gel electrophoresis after PCR. L: 50-bp ladder; 1, 2, 3, 4 shows the 243 bp fragment of MnSOD.
Spontaneous abortion and functional polymorphism (Val16Ala) in the manganese sod gene 3 Table III. Distribution of MnSOD Val16Ala polymorphisms in mono and poly-abortive patients. Mono-abortive
Figure 2. Agarose gel electrophoresis after RFLP Lanes: L, 50-bp ladder; 1, 2 Ala/Ala; 3, 4 Ala/Val; 5, 6 Val/Val.
differ significantly between the mono- and poly-abortive patients (p 0.2) (Table III).
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Discussion To our knowledge, this is the first report evaluating the association between MnSOD Val16Ala polymorphism and the risk of spontaneous abortion. MnSOD is a crucial enzyme counteracting ROS generated in mitochondria. Since the efficient targeting of MnSOD may be an important part of its functional activity and ROS contribute to aetiology of the disease, we performed a case–control study to examine the association between MnSOD genotypes and the risk of spontaneous abortion. The high metabolic rate of placenta leads to increased generation of ROS. As a result, oxidative stress rises during early pregnancy (Safronova et al. 2003). Increased formation of ROS, and even oxidative stress, is considered normal during the 2nd trimester of pregnancy (Wang et al. 1991). However, uncontrolled oxidative stress has been found to be associated with miscarriage during the establishment of cellular and biochemical interactions between the uterine endometrium and the developing post-implanting conceptus (Jenkins et al. 2000). Lyu et al. (2013) found that overproduction of ROS along with downregulating of some genes associated with mitochondrial functions, such as copper/zinc superoxide dismutase (Cu/Zn SOD), would lead to unexplained miscarriage resulting from abnormal responses in placental villi. Adequate placental antioxidant defence may prevent the disorders induced by oxidative stress, including impairment of placental function and abortion (Al-Gubory et al. 2010). The mitochondrial MnSOD is critically required because as much as 90% of cellular ROS can be generated in this organelle. In addition, mitochondrial DNA is particularly susceptible to oxidative damage (Richter et al. 1988). In this study, no significant difference was found between MnSOD Val16Ala polymorphism and the risk of spontaneous abortion. However, more Val/Val genotypes were observed among the patients. Our results were consistent with some previous studies, which found no association Table II. Distribution of MnSOD Val16Ala polymorphisms in women with spontaneous abortion (cases) and women with normal pregnancy history (controls). Controls
Genotype frequency Val/Val Val/Ala Ala/Ala Allele frequency Val Ala
Cases
n
(%)
n
(%)
10 59 10
12.6 74.7 12.6
20 76 4
20.0 76.0 4.0
79 79
50.0 50.0
116 84
58.0 42.0
p value 0.059
0.16
Genotype frequency Val/Val Val/Ala Ala/Ala Allele frequency Val Ala
Poly-abortive
n
(%)
n
(%)
3 21 0
12.5 87.5 0
17 55 4
22.3 72.3 5.2
28 21
56 43
89 63
58 41
p value 0.25
0.9
between MnSOD Val16Ala polymorphism and diseases, such as rheumatoid arthritis (Yen et al. 2002), asthma (Holla et al. 2005) and schizophrenia (Ventriglia et al. 2006). Association studies regarding the MnSOD Val16Ala polymorphism are difficult to interpret. As MnSOD removes the superoxide anion, a potential source of DNA damage, it would be plausible to predict that the MnSOD Val allele would lead to an increased risk of the disease. However, it has not been demonstrated strongly, as several association studies examining the relationship of this polymorphism with diseases, such as breast cancer, had adverse results (Mitrunen et al. 2001; Egan et al. 2003). The role of MnSOD polymorphism on some non-cancerous conditions, such as carotid atherosclerosis (Kakko et al. 2003), Alzheimer (Wiener et al. 2007) and pregnancy rates in IVF (Ruiz-Sanz et al. 2011), also showed contrasting results. In conclusion, no correlation was observed between susceptibility to abortion and MnSOD Val16Ala polymorphism. The effects of MnSOD genotypes may depend on environmental factors that increase or decrease the levels of ROS, and such factors may vary in different populations. Further studies in larger populations are needed to discover the role of this gene polymorphism on spontaneous abortion.
Acknowledgements The authors thank all people in the Genetics and Developmental Biology laboratories, Department of Biology, Faculty of Sciences, University of Guilan, for their technical assistance, and all the sample donors for their collaboration. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This study was partly supported by the University of Guilan.
References Agarwal A, Gupta S, Sekhon L, Shah R. 2008. Redox considerations in female reproductive function and assisted reproduction: from molecular mechanisms to health implications. Antioxidants and Redox Signalling 10:1375–1403. Al-Gubory KH, Fowler PA, Garrel C. 2010. The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. International Journal of Biochemistry and Cell Biology 42:1634–1650. Demetroulis C, Saridogan E, Kunde D, Naftalin AA. 2001. A prospective randomized control trial comparing medical and surgical treatment for early pregnancy failure. Human Reproduction 16:365–369. Egan KM, Thompson PA, Titus-Ernstoff L, Moore JH, Ambrosone CB. 2003. MnSOD polymorphism and breast cancer in a population-based case-control study. Cancer Letters 199:27–33.
J Obstet Gynaecol Downloaded from informahealthcare.com by Universitaet Zuerich on 01/06/15 For personal use only.
4 E. Eskafi Sabet et al. Gupta S, Agarwal A, Banerjee J, Alvarez JG. 2007. The role of oxidative stress in spontaneous abortion and recurrent pregnancy loss: a systematic review. Obstetrical and Gynecological Survey 62:335–347; quiz 353–354. Halliwell B, Gutteridge JM, Cross CE. 1992. Free radicals, antioxidants, and human disease: where are we now? Journal of Laboratory and Clinical Medicine 119:598–620. Hefler LA, Tempfer CB, Bashford MT, Unfried G, Zeillinger R, Schneeberger C et al. 2002. Polymorphisms of the angiotensinogen gene, the endothelial nitric oxide synthase gene, and the interleukin-1beta gene promoter in women with idiopathic recurrent miscarriage. Molecular Human Reproduction 8:95–100. Hempstock J, Jauniaux E, Greenwold N, Burton GJ. 2003. The contribution of placental oxidative stress to early pregnancy failure. Human Pathology 34:1265–1275. Holla LI, Kankova K, Vasku A. 2005. Functional polymorphism in the manganese superoxide dismutase. Clinical Biochemistry 39:299–309. Homer H, Li T, Cooke I. 2000. The septate uterus: a review of management and reproductive outcome. Fertility and Sterility 73:1–14. Jenkins C, Wilson R, Roberts J, Miller H, McKillop JH, Walker JJ. 2000. Antioxidants: their role in pregnancy and miscarriage. Antioxidants and Redox Signalling 2:623–628. Kakko S, Paivansalo M, Koistinen P, Kesaniemi YA, Kinnula VL, Savolainen MJ. 2003. The signal sequence polymorphism of the MnSOD gene is associated with the degree of carotid atherosclerosis. Atherosclerosis 168:147–152. Lyu SW, Song H, Yoon JA, Chin MU, Sung SR, Kim YS et al. 2013. Transcriptional profiling with a pathway-oriented analysis in the placental villi of unexplained miscarriage. Placenta 34:133–140. McDonald AD, Armstrong BG, Sloan M. 1992. Cigarette, alcohol, and coffee consumption and prematurity. American Journal of Public Health 82:87–90. Mitrunen K, Sillanpaa P, Kataja V, Eskelinen M, Kosma VM, Benhamou S et al. 2001. Association between manganese superoxide dismutase (MnSOD) gene polymorphism and breast cancer risk. Carcinogenesis 22:827–829. Richter C, Park JW, Ames BN. 1988. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proceedings of the National Academy of Sciences of the United States of America 85:6465–6467. Rosenblum J, Gilula N, Lerner R. 1996. On signal sequence polymorphisms and diseases of distribution. Proceedings of the National Academy of Sciences of the United States of America 93:4471–4473. Ruiz-Sanz JI, Aurrekoetxea I, Matorras R, Ruiz-Larrea MB. 2011. Ala16Val SOD2 polymorphism is associated with higher pregnancy rates in in vitro fertilization cycles. Fertility and Sterility 95:1601–1605.
Ruutiainen K, Seppala M. 1991. Polycystic ovary syndrome: evolution of a concept. Current Opinion in Obstetrics and Gynecology 3:326–335. Safronova VG, Matveeva NK, Avkhacheva NV, Sidel’nikova VM, Van’ko LV, Sukhikh GT. 2003. Changes in regulation of oxidase activity of peripheral blood granulocytes in women with habitual abortions. Bulletin of Experimental Biology and Medicine 136:257–260. Sarig G, Younis J, Hoffman R. 2002. Thrombophilia is common in women with idiopathic pregnancy loss and is associated with late pregnancy wastage. Fertility and Sterility 77:342–347. Shimoda-Matsubayashi S, Matsumine H, Kobayashi T, Nakagawa-Hattori Y, Shimizu Y, Mizuno Y. 1996. Structural dimorphism in the mitochondrial targeting sequence in the human manganese superoxide dismutase gene. A predictive evidence for conformational change to influence mitochondrial transport and a study of allelic association in Parkinson’s disease. Biochemical and Biophysical Research Communications 226:561–565. Sutton A, Khoury H, Prip-Buus C, Cepanec C, Pessayre D, Degoul F. 2003. The Ala16Val genetic dimorphism modulates the import of human manganese superoxide dismutase into rat liver mitochondria. Pharmacogenetics 13:145–157. Todorova K, Ivanov S, Mazneikova V, Genova M. 2005. Glucooxidative stress and spontaneous abortion in pregnant women with diabetes mellitus type 1. Akusherstvo i Ginekologiia 44:3–10. Ventriglia M, Scassellati C, Bonvicini C, Squitti R, Bevacqua MG, Foresti G et al. 2006. No association between Ala9Val functional polymorphism of MnSOD gene and schizophrenia in a representative Italian sample. Neuroscience Letters 410:208–211. Vural P, Akgul C, Yildirim A, Canbaz M. 2000. Antioxidant defence in recurrent abortion. Clinica Chimica Acta 295:169–177. Wan XS, Devalaraja MN, ST Clair DK. 1994. Molecular structure and organization of the human manganese superoxide dismutase gene. DNA and Cell Biology 13:1127–1136. Wang YP, Walsh SW, Guo JD, Zhang JY. 1991. Maternal levels of prostacyclin, thromboxane, vitamin E, and lipid peroxides throughout normal pregnancy. American Journal of Obstetrics and Gynecology 165:1690–1694. WHO. 1976. World Health Organization recommended definitions, terminology and format for statistical tables related to the perinatal period and use of a new certificate for cause of perinatal deaths. Acta Obstetricia et Gynecologica Scandinavica 56:247–253. Wiener HW, Perry RT, Chen Z, Harrell LE, Go RC. 2007. A polymorphism in SOD2 is associated with development of Alzheimer’s disease. Genes, Brain, and Behavior 6:770–775. Yen JH, Chen CJ, Tsai WC, Lin CH, Ou TT, Hu CJ et al. 2002. Manganese superoxide dismutase and cytochrome P450 1A1 genes polymorphisms in rheumatoid arthritis in Taiwan. Human Immunology 64:366–373.
